We propose to determine the role of proteolytic enzymes in normal lens development and in cataractogenesis. Characterization of bovine lens neutral proteinase, a major endopeptidase activity of the lens, will precede our study of proteolysis in the human lens. Based on our determination of neutral proteinase specificity for cleavage at aliphatic amino acid residues, a synthetic substrate for this enzyme has been identified. Utilizing hydrolysis of this chromogenic substrate to assay activity, an alternative, more complete purification procedure will be pursued. Subsequently we will determine the kinetics, reaction mechanism and molecular properties of the enzyme. Analysis of products of hydrolysis of purified Alpha-crystallin peptides by neutral proteinase will relate the in vitro activity to degraded peptides observed in vivo. Differences in endopeptidase and exopeptidase activities in galactose-induced cataract as compared to normal levels in the rat will be determined using chromogenic and fluorogenic synthetic substrates. An in vitro culture system will be used to directly measure protein degradation using a double-label isotope technique under conditions which induce or prevent cataract formation. Proteolytic enzyme activities will be measured in lenses incubated under these same conditions. The possibility of modifying the extent of degradation and cataract formation by manipulation of the culture medium will be explored. The findings from the work with animal model systems will be applied to a study of the human lens. We will compare proteolytic enzyme activity in the normal and cataractous human lens, as well as purify and characterize the human lens neutral proteinase. If proteolysis is involved in cataractogenesis, control of degradative activity may be an alternative target for prevention or retardation of lens opacification.